8  Allelopathy Methods

8.1 Bioassay set up

Even through you are working as part of a group, each of you will set up a control and a treatment assay, essentially these become replicates as part of your experimental design and you will combine your data across replicates to get a larger sample size and minimize effects of uncontrolled variables/confounding factors. Make sure that you read through the instructions before you get started and discuss with your group how you will implement them. Make sure that you keep communicating with your lab partners as you set up your experiment to make sure that you are being as consistent as possible.

  1. Obtain two complete petri dishes. Double check that you have a matching top and bottom; when resting on a surface, the open rim of the bottom part of the dish should rest against the underside of the overlapping lid of the dish. If this is not the case, swap tops and bottoms or get a new set of dishes.
  2. Cut four pieces of paper towel that will fit into the bottom of a petri dish. Then place two pieces of paper towel into the bottom of each of your two petri dishes. 
  3. Label the tops of both dishes with your name, section, and treatment (“Exp” or “Ctrl”). 
  4. Finely chop/mince your plant matter on a cutting board. 
  5. Use weigh boats to weigh out 0.8gof plant material.
  6. Use the tin foil to make a small container for the plant material, then place the aluminum container with the plant material into the experimental dish so the plant material will never contact the water in the dish, but is free to release volatile chemicals into the dish atmosphere. Make sure the you are using the same amount of tin foil and configuration in your experimental and control dish and your group members are doing the same. 
  7. Wash your hands really well to remove any chemicals picked up on your fingertips from the chopped up plant matter. Then obtain 40 radish seeds using clean small weigh boat. Place 20 of the seeds on the paper towel (not in tin foil!) in your experimental dish and 20 seeds on the paper in your control dish. Spread the seeds out with the tip of a pen or pencil or scissors. Communicate with your group mates to standardize your set up.
  8. Open the dishes, wet the paper in each Petri dish with ~10 ml of distilled water using a small beaker. Make sure you are being consistent in the amount of water you and your group mates are using.
  9. Place your dishes on a tray.
  10. Carefully wash knives, presses, cutting boards, weighing boats and small petri dishes and set out to dry on paper towels next to sinks. Make sure everything is rinsed well.

8.2 Determine germination rate

You will need to come into the lab approx. 48 hours after you start the experiment. Your data should be collected no later than 60 hours after your initial set up. Ideally, each of you will count their own seeds, if there is an issue coming in to count seeds due to work/class schedule etc. please make sure you communicate this to your lab group and somebody else is able to count your seeds. You are responsible to make sure you remind them. If your germination rate for the entire group is not determined at approx. the same time you will not be able to analyze your data set properly.

  1. Open the GoogleSheet to record your data and and navigate to the tab for your lab group. Familiarize yourself with the data sheet and how you will enter your data.
    • Column A: enter the name of your plant you are testing; this will be the same for all of your group members1.
    • Column B: Recall that the petri dishes set up by each group member are replicates that help us control for confounding variables and increase our sample size. Enter your lastname.
    • Column C: Here you will record your treatment, choose either “ctrl” or “exp” from the dropdown menu as you enter information from your Control and Experiment petri dish respectively.
    • Column D: We are going to arbitrarily number your seeds 1 - xx to distinguish them in the data set. You do not have to be able to associate the numbers back to your seeds.
    • Column E: As you check each seed chose either “germinated” or “no germination” from the menu.
    • Column F: Record the date you set up your assays. This will be the same for all of your data points.
    • Column G: Record the time you set up your assays. This doesn’t have to be exact to the minute, within an hour is fine.
    • Column H: Record the date you came in to record the germination status.
    • Column I: Record the time you finished recording your data2.
  2. Recover and open your first petri dish. Inspect it to see if there are any concerns about your assays, e.g., excessive drying, leaky boats, etc.; if so take a picture for your records and notify your instructor. Record whether you are checking seeds in a Control or Experiment dish in your datasheet.
  3. Check each seed and record it as having germinated if it has a visible radicle3. You may need to move seeds around a bit4 to see if they truly have germinated. Do NOT measure the length of seeds at this time.
  4. Repeat the process with your second dish.
  5. Double check your data entry to make sure you have included all the information (see point 1).

  • 1 This might seem unnecessary but is an example of building good data management habits that can save a lot of work and headaches down the line. Including it means that you could combine data with other groups at a later point and that way the data set itself includes this information if that information should be lost down the line.

  • 2 The reason why we are including this information on when the assays were set up and when you recorded your observations is that if your results are unusual or inconsistent you would be able to go. Frequently, scientists are working on multiple projects or you might have several people collaborating and data might not be analyzed right after it is recorded. Consider “future you” as your most important collaborator and keeping good records is being a good collaborator.

  • 3 This is the “primary” or “seed” root.

  • 4 A pencil or similar is helpful.

    • 8.3 Determine seedling lengths

    We will determine seedling length for all seeds that germinated one week after set-up during our normal lab period.

    1. Open the GoogleSheet to record your data and and navigate to the tab for your lab group. Familiarize yourself with the data sheet and how you will enter your data.
      • Column A: Enter the name of your plant you are testing.
      • Column B: Enter your last name to distinguish your replicates from those of your group members.
      • Column C: Choose either “ctrl” or “exp” from the dropdown menu as you enter information from your Control and Experiment petri dish respectively.
      • Column D: We are going to arbitrarily number your seedlings 1 - xx to distinguish them in the data set. You don’t have to be able to associate the numbers back to your seedlings.
      • Column E: We will record the length for all germinated seeds.
    2. Retrieve your petri dishes with your seeds. Inspect them to see if there are any concerns about your assays, e.g., excessive drying, leaky boats, etc. If this is the case call your instructor over to inspect. If there are any issues make sure to take notes that you can refer to when you discuss your results.
    3. Retrieve a small ruler to measure the length of your seedlings. Start with your control dish. Gently separate your seedlings and lay them out on a piece of paper. Straighten each seedling as much as you can and then measure the length. You may have to break or cut some seedlings to straighten them. Even if only a small amount of growth has occurred still measure and include those values. Record lengths in the Google Sheet in millimeters. In some cases, only a small bit of growth may have occurred (only a few mm of growth may be have occurred)5.
    4. Repeat Step 2 for your experimental dish.
    5. When you are finished, double check that you measured all your seedlings and recorded all your data. Then dump your paper towel and seedlings in the trash along with your tin foil boats and plant material. Rinse your petri dishes and set them up to dry.

  • 5 It is possible that some seeds that had not germinated when you first checked them have now germinated. Do not change your original germination data. We want to know how many seeds germinated within about 48-60 hours!